The integrins are a family of integral membrane receptors which mediate cell-cell and cell-extracellular matrix interaction by regulating cell adhesion, differentiation, migration and the immune response. The integrins are heterodimers composed of α and β subunits. Different combinations of subunits are expressed by different cell types generating a family of 25 different heterodimers. Some integrin receptors from the RGD binding subgroup, such as the α5β1 and the αvβ3 integrin receptors, recognize the arginine-glycine-aspartic acid (RGD) motif in extracellular matrix proteins such as fibronectin. Peptides comprising the RGD motif or structural mimics thereof have been prepared and proposed as therapeutic entities binding to integrins, primarily the α5β1 and the αvβ3 integrin receptor, thereby inhibiting the integrin-mediated cell attachment to extracellular matrix proteins such as fibronectin (cf. U.S. Pat. No. 5,536,814; U.S. Pat. No. 5,627,263; U.S. Pat. No. 5,817,750; U.S. Pat. No. 5,955,572). These peptides are proposed for use in wound healing and to prevent tumour cells from binding to fibronectin, thereby inhibiting metastasis. By binding integrin receptors expressed on proliferating endothelial cells, these peptides can improve the drug delivery during cancer progression and inflammation (G. A. Konning et al., Arthritis Rheum 54 (4) 2006 pp 1198-1208 and X. B. Xiong et al., J Pharm Sci 94 (8) 2005, pp 1782-1793).
The integrin α2β1 receptor belongs to the integrin subgroup of collagen binding receptors. It binds several naturally occurring ligands such as collagen I, through the extracellular I domain of the integrin α2 monomer (the aα2 I domain), which is conserved in all collagen binding integrin receptors (M. Barczyk et al., Cell tissue Res 339 (1) 2010 pp 269-280). The interaction occurs in a cation-dependent manner, the interaction between collagen I and the integrin α2β1 receptor being mediated via Mg2+-bridged interactions supported by the MIDAS motif residues (Asp 151, Ser 153, Thr 221 and Asp 254) (L. J. Lambert et al., J. Biol. Chem. 283 (24), 2008, pp. 16665-16672). Solving the structure of the integrin α2 domain in complex with a collagen triple helix of three 21 amino acid collagen-derived peptides revealed that the α2 I domain undergoes conformational changes upon interaction with collagen (Emsley et al., Cell. 101 (1), 2000, pp 47-56). Such conformational changes in the receptor on interaction with the collagen peptide were confirmed by NMR spectroscopy (Lambert, supra). The presence of the triplex peptide also inhibits interaction between the integrin α2 and full-length collagen (Knight et. al., J. Biol. Chem. 275 (1), 2000, pp 35-40).
Binding of collagen and fibrinogen-dependent platelet activity may be inhibited by polypeptide toxins derived from snake venom, contributing to the anticoagulant effect of these venoms. These polypeptides (termed disintegrins) are functional homologues of the RGD motif found in extracellular matrix proteins. The jararhagin protein isolated from the venom of Bothrops jararaca (the Brazilian pit viper) has been found to inhibit collagen I interaction (J. Ivaska et al., J. Biol. Chem. 274 (6), 1999, pp. 3513-3521). A basic motif, Arg-Lys-Lys (RKK), in jararhagin is important for the interaction. A nine amino acid long cyclic peptide (C241TRKKHDNAQ249C)(SEQ ID NO: 3) has been found to inhibit the interaction between collagen I and the integrin α2β1 receptor in the presence of 2 mM MgCl2 (Ivaska et al., supra) by binding competitively to α2 I domain and disrupting cell adhesion to collagen I.
The nine amino acid peptide containing the RKK motif disclosed by Ivaska et al., supra, and other cyclic peptides containing the RKK motif derived from jararhagin are disclosed in WO 99/02551 and proposed for use in therapy to block integrin interaction with collagen and laminin, more specifically to block cell migration on collagen, e.g. as seen in periodontitis, or to block migration of malignant cells, e.g. as seen in osteosarcoma or malignant melanoma, or to prevent platelet adhesion to collagen, e.g. as seen in thrombosis and stroke. The peptides disclosed in WO 99/02551 preferably contain a cysteine residue at both ends such that the peptide may be cyclized by disulfide bond formation between the two cysteines.
The integrin α2β1 receptor has been found to be expressed in high amounts in skin keratinocytes (F. Watt, EMBO J., 21 (15), 2002, pp 3919-3926). In normal skin, the receptor is confined to the proliferating basal layers of the epidermis, but during wound healing and in psoriasis, the receptor is expressed on keratinocytes in the suprabasal layers correlating with an altered keratinocyte differentiation. Transgenic mice expressing the integrin α2β1 receptor under the involucrine promoter have previously been demonstrated to spontaneously develop a skin disorder resembling psoriasis (J. M. Carroll et al., Cell 83, 1995, pp. 957-968). It has furthermore been shown that in integrin transgenic mice a mild epidermal wounding leads to chronic inflammation similar to the Koebner phenomenon in psoriasis patients in whom wounding of non-lesional skin often results in the development of a psoriatic plaque at the site of the wound. The mice were followed for five weeks and throughout that time exhibited substantial keratinocyte hyperproliferation, inflammatory infiltration and high cytokine levels within the skin. Furthermore, the systemic immune response was very much affected with increased spleen size, elevated cytokine levels in serum and altered lymphocyte trafficking resembling what is seen in psoriasis patients (I. Teige et al., Int. Immunopharmacol. 10, 2010, pp. 107-114).
It is an object of the invention to provide a dermal drug delivery system utilizing integrin receptors expressed on keratinocytes in the lower epidermis as well as on inflammatory cells present in skin to target pharmacologically active compounds to this specific layer of the skin with a view to treating dermal diseases located in the lower epidermis (e.g. psoriasis, wounds, skin cancer and atopic dermatitis).